Early detection of diseased cells by molecular imaging is viewed as the best hope by many in treating cancer and other diseases. By using agents that attach selectively to diseased cells, the ability to image disease states at the cellular level is further enhanced. This affinity may be achieved through the use of targeting agents which seek receptors or biomarkers in diseased cells. As a result of these advances there has been a significant acceleration of research, leading to an increased need for safe and effective targeted molecular imaging agents (TMIAs) for MRI, PET, SPECT, PAI, and NIR fluorescence imaging and combinations of those. Dual modal TMIAs can include agents for MRI-PET, MRI-FLA, and MRI-PAI. Tri-modal agents may also be combined in one agent.
In conventional imaging agent synthesis, one in which a metal is incorporated into DOTA or related chelating groups like DTPA, DO3A, TE2A. NOTA, CB-TETA, a metal such as Gd (for MRI) or radioactive metals such as Tc, In, Ga and Cu (for PET or SPECT) are typically inserted during the final steps of a synthesis, regardless of whether the agent is a peptide, protein, antibody, nanobody, or large assembly such as a dendrimers, polymers, nanoparticles, or small molecules. It is most common in a multi-step synthesis to bring in a DOTA or DTPA, for example, in the second to last step and introduce Gd or the radioactive metal as the last step. This is because DOTA contains four amines and four acids which are problematic in chemical reactions like peptide coupling.
In the synthesis of imaging agents a common solution is to bring a t-butyl ester protected form of DOTA and to carry this fully protected chelating group through the synthesis. An example is the elaboration of peptide based imaging agents utilizing a tri-t-butyl ester of DOTA on the side chain of lysine as a starting material (Leun-Rodriguez, et al). Fully protected DOTA precursors are commercially available containing t-butyl esters on the multi acid groups. There are no commercially available alternative protecting groups apart from this form. The removal of t-butyl groups requires strong acids like TFA, and many peptides, proteins, antibodies, and other groups on imaging agents such as dyes, and in particular many NIR dyes, may not be stable to such harsh conditions.
Another method of introducing DOTA, DTPA and other groups is to react an activated form of the unprotected chelating ligand (NHS ester or isothiocyanate are examples) directly, followed by insertion of the metal, such as Gd. Very few chemical steps such as coupling or conjugation of additional targeting groups or imaging agents can occur in the presence of the unprotected, non-chelated DOTA because the acids are reactive. In addition, it is difficult to purify intermediates containing the multiple unprotected acid and amine groups.
These restrictions severely limit the way in which DOTA and metal-DOTA complexes for MRI and radioactive metals for PET can be incorporated into new imaging agents. It would therefore be very useful to have an alternative way to add chelating groups containing metals, to allow practical synthetic approaches to new types of imaging agents.
There are few methods of combining various dyes for use in NIRF or PAI imaging in a straightforward and easily applicable manner. Likewise, there are few generally applicable methods for combining dyes and metal-chelating complexes into the same imaging agent while also providing a method for conjugating targeting groups as a final step.
It would be therefore useful to provide routes to a wide variety of targeted molecular imaging agents (TMIAs) by providing imaging agents comprised of pre-formed dyes and metal chelates, either alone or in combination, in a form that could be attached or conjugated to any targeting agent containing a reactive amine, sulfide or carboxylic acid by direct conjugation or by means of a well-established linker.